Molecularly targeted therapies directed against signaling pathways that control cell growth, proliferation and differentiation in malignant lymphoblasts have recently emerged as promising tools in the therapy of human leukemias. In this regard, the identification of activating mutations in the NOTCH-1 receptor in over 50% of human T-cell acute lymphoblastic leukemias (T-ALL) has prompted the initiation of clinical trials to test the effectiveness of drugs inhibiting NOTCH-1 signaling in this disease.
The NOTCH signaling pathway is a critical regulator of cell fate, specification, and stem cell homeostasis in the hematopoietic system. There are three fundamental components of the NOTCH pathway: the DSL ligands (Delta-like 1, 3 and 4; and Jagged 1 and 2), the NOTCH receptors (NOTCH-1-4), and the CSL DNA binding protein, a transcription factor that interacts with the activated form of NOTCH receptors and mediates the conversion of NOTCH-activating signals at the cell surface into changes in gene expression in the nucleus.
Mature NOTCH receptors are generated from a precursor polypeptide that is post-translationally cleaved into two fragments by a furin protease during its maturation in the trans-Golgi network. In the resting receptor, these two fragments interact to form a heterodimeric transmembrane protein. Upon binding to its ligands, the transmembrane portion of NOTCH-1 is sequentially cleaved first by an ADAM protease and then by the γ-secretase complex. This final endomembrane cleavage step releases the active intracellular fragment of NOTCH-1 (ICN1), which translocates to the nucleus and activates target gene expression by forming a ternary complex with the CSL DNA-binding protein and the MAML1 transcriptional coactivator. Importantly, the presenilin γ-secretase complex also has a pathogenic role in Alzheimer's disease, fostering the development of highly active γ-secretase inhibitors (GSIs) for the treatment of this neurodegenerative disease.
GSIs effectively inhibit the last proteolytic cleavage required for the activation of the NOTCH-1 receptor and have been shown to induce cell cycle arrest in T-ALL cell lines in vitro. The cytostatic effects of GSIs seem to be mediated by the down regulation of a transcriptional regulatory network controlling macromolecular metabolism, cell growth and proliferation downstream of NOTCH-1 in T-ALL. Activation of NOTCH receptors has also been implicated in the pathogenesis of numerous solid tumors, including breast and ovarian carcinomas and medullobastoma, thereby supporting a possible role for GSI's in the treatment of solid tumors.
Glucocorticoids (GCs) are a group of bioactive molecules capable of binding the glucocorticoid receptor (GR) and encompass cortisol, a natural hormone, and a number of structurally related compounds. In resting conditions, the glucocorticoid receptor is located in the cytosol, in close association with inactivating heat shock proteins. Binding to glucocorticoids induces conformational changes that release the GR from heat shock proteins, induce its dimerization and promote translocation to the nucleus where it binds to DNA and regulates the expression of target genes. In addition to this direct role as a ligand-activated transcriptional regulator, the GR also affects gene expression by inhibiting the activity of other transcription factors such as AP1 and NFκB.
Physiologic glucocorticoid signaling plays important roles in the regulation of immune responses and the generation of the immune repertoire. However, pharmacologic doses of glucocorticoids induce cell cycle arrest and apoptosis in normal lymphocytes and have direct anti-cancer activity against lymphoid malignancies. Indeed, glucocorticoids have been used in the treatment of lymphoid tumors since the early days of chemotherapy and constitute part of the core treatment for acute lymphoblastic leukemia (ALL). The response rates to glucocorticoid monotherapy in primary pediatric acute lymphoblastic leukemia range between 45 and 65%. However, after relapse the rate of subsequent remission induction with glucocorticoids alone falls to 25%. The importance of glucocorticoids in the treatment of ALL is emphasized by the excellent prognosis associated with in vivo early response to glucocorticoid therapy. In contrast, ALL patients whose lymphoblasts show in vitro resistance to glucocorticoid-induced apoptosis have a less favorable prognosis.
Although the specific mechanisms that mediate glucocorticoid induced cell death and glucocorticoid resistance are not fully understood, several lines of evidence support that the mitochondrial/intrinsic cell death pathway mediates glucocorticoid induced apoptosis.
Activation of the GR induces the expression of the pro-apoptotic BH3-only gene BIM in ALL cells and both BIM and PUMA, a second pro-apoptotic BH3-only factor, are necessary for appropriate GC-induced apoptosis. Conversely, lymphocytes from double knockout Bax−/− Bak−/− mice, which have a complete block in the intrinsic apoptotic pathway, are resistant to glucocorticoid-induced apoptosis. Finally, high-level expression of the anti-apoptotic factor MCL1 has been correlated with glucocorticoid resistance in vitro, and turning the balance of pro-apoptotic and anti-apoptotic factors towards cell survival by BCL2 or MCL1 overexpression can protect lymphoblastic leukemia cells from GC-induced programmed cell death.
Enthusiasm for GSIs in the treatment of T-ALL, however, is often tempered by the apparent inability of these drugs to induce robust cytotoxic effects towards human leukemic lymphoblasts as single agents. In addition, others have shown that aberrant NOTCH-1 can antagonize glucocorticoid-induced cell death in normal developing thymocytes. Still further, severe gastrointestinal toxicity limits the clinical application of GSIs. Accordingly, a need exists for methods and compositions that enhance the efficacy of, mitigate resistance to, and reduce the gut toxicity of GSIs in the treatment of T-ALL and other conditions, such as Alzheimer's disease.